Detection of TMPRSS2-ERG fusion gene in benign prostatic hyperplasia.

The Ets-related gene fusions are among the most common molecular alterations in prostate cancer (PCa) and are detected in more than 50 % of PCas. Transmembrane protease serine 2 and Ets-related gene fusion (TMPRSS2-ERG) is the most frequently identified chimeric gene and has been associated with undifferentiated and invasive phenotypes. TMPRSS2-ERG has also been detected in prostate intraepithelial neoplasia (PIN) lesions and more rarely in benign prostatic hyperplasia (BPH) regions mainly in PCa-bearing glands. The possibility that the fusion TMPRSS2-ERG may be present in BPH samples in the absence of apparent PCa was addressed. Out of 115 BPH samples, three were found positive employing RT-PCR. The presence of the fusion gene was confirmed by FISH for these samples, and an additional four samples were found to carry the TMPRSS2-ERG fusion out of 43 tested by the later approach. The presence of the TMPRSS2-ERG fusion did not result in altered expression of 12 putative downstream targets. These findings indicate that TMPRSS2-ERG may or may not lead to PCa development.

Curcumin analogues as possible anti-proliferative & anti-inflammatory agents.

A series of novel curcumin analogues has been designed, synthesized and tested in vitro/in vivo as potential multi-target agents. Their anti-proliferative and anti-inflammatory activities were studied. Compounds 1b and 2b were stronger inhibitors of soybean lipoxygenase (LOX) than curcumin. Analogue 1b was also the most potent aldose reductase (ALR2) inhibitor. Two compounds, (1a and 1f) exhibited in vivo anti-inflammatory activity comparable to that of indomethacin, whereas derivative 1i exhibited even higher activity. The derivatives were also tested for their anti-proliferative activity using three different human cancer cell lines. Compounds 1a, 1b, 1d and 2b exhibited significant growth inhibitory activity as compared to curcumin, against all three cancer cell lines. Lipophilicity was determined as R(M) values using RPTLC and theoretically. The results are discussed in terms of the structural characteristics of the compounds. Docking simulations were performed on LOX and ALR2 inhibitor 1b and curcumin. Compound 1b is well fitted in the active site of ALR2, binding to the ALR2 enzyme in a similar way to curcumin. Allosteric interactions may govern the LOX-inhibitor binding.

mRNA quantification and clinical evaluation of telomerase reverse transcriptase subunit (hTERT) in intracranial tumours of patients in the island of Crete.

Telomerase is a reverse transcriptase that maintains telomeres by adding telomeric TTAGGG repeats to the ends of human chromosomes. The aim of this study was to evaluate quantitatively the mRNA expression of telomerase catalytic subunit (hTERT) in different types of intracranial tumours in relation to their histologic pattern and grade and correlate it with progression-free (PFS) and overall survival (OS) of patients. Human telomerase reverse transcriptase mRNA levels were estimated by the use of real time RT-PCR in 68 samples of intracranial tumours. It revealed statistical correlation between hTERT mRNA expression levels and the grade of the tumours (P<0.001). Patients having negative expression of hTERT mRNA had statistically longer PFS (P=0.031) and OS (P=0.047). Cox univariate regression analysis revealed that hTERT mRNA-positive patients had a high and statistically significant risk of relapse (hazard ratio (HR) of 2.24 and P=0.038). In the Cox multivariate regression model, the levels of hTERT mRNA were adjusted for tumour grade and patients age, and since there was statistically significant relationship between the levels of hTERT mRNA and the grade of the tumours (P=0.003 or P=0.006, respectively), hTERT mRNA levels could not be considered as an independent prognostic factor for PFS or OS.

CRD-BP: a c-Myc mRNA stabilizing protein with an oncofetal pattern of expression.

The Coding Region Determinant-Binding Protein (CRD-BP) is an RRM and KH-domain-containing protein that recognizes specifically at least three RNAs. It binds to one of the two c-myc mRNA instability elements, to the 5'Un Translated Region (UTR) of the leader 3 IGF-II mRNA and to the oncofetal H19 RNA. CRD-BP has been assigned a role in stabilizing c-myc mRNA by preventing its endonucleolytic cleavage and in repressing the translation of the leader 3 IGF-II mRNA, the major embryonic species of this message. CRD-BP is normally expressed only in fetal tissues. However, its expression is detected in primary tumors and transformed cell lines of different origins. The vast majority of colon (80%) and breast (60%) tumors and sarcomas (73%) express CRD-BP whereas in other tumor types, for example prostate carcinomas, its expression is rare. CRD-BP expression has also been detected in benign tumors such as breast fibroadenomas, meningiomas and other benign mesenchymal tumors, implying a role for this gene in abnormal cell proliferation. In breast carcinomas, CRD-BP expression and or gene copy number gains in the region encompassing the c-myc locus were detected in approximately 75% of tumors, implying that the deregulated expression of c-myc may be more widespread than previously believed. Infiltrated lymph nodes, corresponding to CRD-BP-positive primary tumors, were also found positive indicating that monitoring for CRD-BP could prove useful for the detection and monitoring of disseminated disease.

C-MYC and IGF-II mRNA-binding protein (CRD-BP/IMP-1) in benign and malignant mesenchymal tumors.

Mouse coding region determinant-binding (mCRD-BP) and human IGF-II mRNA-binding 1 (hIMP-1) proteins are orthologous mRNA-binding proteins that recognize c-myc and IGF-II mRNA, respectively, and regulate their expression posttranscriptionally. Here, we confirm that human CRD-BP/IMP-1 binds to c-myc mRNA and that it is predominantly expressed in fetal tissues. Moreover, hCRD-BP/IMP-1 expression was detected in cell lines of neoplastic origin and in selected primary tumors. In a series of 33 malignant and 10 benign mesenchymal tumors, 73% and 40%, respectively, were found to express hCRD-BP/IMP-1. In particular, expression was significant in 14 Ewing's sarcomas, all of which were positive. The data suggest that hCRD-BP/IMP-1 plays a role in abnormal cell proliferation in mesenchymal tumors.

Overexpression of matrix-metalloproteinase-9 in human breast cancer: a potential favourable indicator in node-negative patients.

Matrix metalloprotease-9 (MMP-9; 92 kDa type IV collaganase, gelatinase B) is regarded as, important for degradation of the basement membrane and extracellular matrix during cancer invasion and other tissue-remodelling events. In this study we evaluate the prognostic value of MMP-9, by immunoperoxidase staining in a series of 210 breast cancer tissues. The results were quantitated using the HSCORE system, which consider both staining intensity and the percentage of cells stained at given intensities. MMP-9 status was compared with the concentration of cytosolic Cathepsin-D and with other established prognostic factors, in terms of disease free survival and overall survival. The median follow-up period was 62 months. MMP-9 staining was observed primarily in cancer cells, and to a lesser degree in surrounding stromal cells. MMP-9 expression was not detected in normal breast tissue. Levels of MMP-9 expression below the cut-off point were more frequently observed in larger (P = 0.014), invasive ductal histologic (P = 0.037), progesterone receptor (PR)-negative and PR-strong positive tumours (P< 0.001), as well as samples belonging to patients with stage III-IV disease (P = 0.009) and age 45-55 years (P = 0.011). In univariate analysis, node-negative breast cancer patients with tumors positive for MMP-9 had a considerable reduction in risk for relapse (RR = 0.45;P = 0.039) or death (RR = 0.32;P = 0.009). Multivariate analysis indicated that MMP-9 status was an independent favourable predictor of OS (RR = 0.47;P = 0.034) in node-negative but not in node-positive patients. Our results suggest that MMP-9 may be an independent favourable prognostic factor in node-negative breast cancer patients. The overexpression of MMP-9 in breast cancer may be also used as a marker to subdivide node negative breast cancer patients in order to determine the optimal treatment modality.

Labdane type diterpenes down-regulate the expression of c-Myc protein, but not of Bcl-2, in human leukemia T-cells undergoing apoptosis.

Sclareol (1) and ent-3beta-hydroxy-13-epi-manoyl oxide (2) belong to the labdane type diterpenes. They were isolated from the leaves and from the fruits of Cistus creticus subsp. creticus, and were found to be active against human leukemic cell lines. Compound 2 was converted to its thiomidazolide derivative (3). Compounds 1 and 3 were found to induce apoptotic cell death in human T-cell leukemia lines and to interfere with their cell cycle, arresting cells at G(0/1) phase. Apoptosis can involve the activation and/or suppression of critical genes such as c-myc whose reduction or its inappropriate expression can be associated with induction of cell death and bcl-2 whose activation prevents apoptosis in the latter case. In order to detect any concomitant effect (1 and 3) upon c-myc and bcl-2 oncogene expression, we performed Western blot analysis to determine the levels of expression of these two genes upon treatment with the above compounds. Western blot analysis showed that of c-myc proto-oncogene levels were markedly reduced before massive apoptosis ensued in H33AJ-JA1 and MOLT3 cells, while bcl-2 expression remained unaffected. Thus, induction of apoptosis due to compounds 1 and 3 in these T-cell leukemic cell lines is preceded by c-myc down regulation and furthermore sustained bcl-2 expression does not rescue cells from apoptosis under the conditions used.

Polyadenylate polymerase enzymatic activity in mammary tumor cytosols: A new independent prognostic marker in primary breast cancer.

Polyadenylate polymerase (PAP) is one of the enzymes involved in the formation of the polyadenylate tail of the 3' end of mRNA. High levels of PAP activity were associated with rapidly proliferating cells. Here we evaluate the prognostic value of PAP activity in breast cancer patients. PAP specific activity values were measured by a highly sensitive assay in the tumor cytosols of 228 women with primary breast cancer. The median follow-up period was 58 months. PAP specific activity values ranged from 2.1-39.4 units/mg protein in the breast tumor cytosols, and the activity was correlated with the level of expression of the antigen. An optimal cutoff value of 5.5 units/mg extracted protein was first defined by statistical analysis. PAP status was then compared with other established prognostic factors in terms of relapse-free survival (RFS) and overall survival (OS). PAP activity levels had a tendency to increase with tumor-node-metastasis (TNM) stage and were higher in node-positive patients. Evaluation of the prognostic value of PAP was performed using univariate and multivariate analyses. Univariate analysis showed that PAP-positive patients had a less favorable prognosis for both RFS (relative risk (RR) = 2.35; P < 0.001] and OS (RR = 3.15; P < 0.001). PAP significantly added to the prognostic power for RFS (RR = 2.51; P = 0.0012) and OS (RR = 4.21; P < 0.001) in multivariate analysis, whereas patient age, tumor size, and nodal and ER status remained independent factors for predicting survival. When only node-negative patients were examined, PAP was found to be an independent factor for predicting RFS (RR = 3.68; P = 0.0032) and OS (RR = 4.81; P < 0.001). PAP did not appear to have a prognostic significance for node-positive patients. PAP is a new prognostic factor for early recurrence and death in breast cancer patients. Our results suggest that PAP may be used as an independent unfavorable prognostic factor in node-negative breast cancer patients because there were no significant associations between PAP and the other prognostic indicators evaluated in this group of patients.

Determination of c-myc amplification and overexpression in breast cancer patients: evaluation of its prognostic value against c-erbB-2, cathepsin-D and clinicopathological characteristics using univariate and multivariate analysis.

C-myc and c-erbB-2 amplification and/or overexpression as well as total cathepsin-D (CD) concentration have been reported to be associated with poor prognosis in breast cancer. The prognostic significance, however, remains somewhat controversial, partly because of discrepancies among the different methodologies used. We determined the amplification and overexpression of c-myc oncogene in 152 breast cancer patients and examined its prognostic value in relation to c-erbB-2 amplification and overexpression, high concentration of CD (> or = 60 pmol mg(-1) protein) and standard clinicopathological prognostic factors of the disease. High CD concentration, as well as c-myc amplification and overexpression, proved to be the best of the new variables examined for prediction of early relapse (ER; before 3 years). After multivariate analysis only CD remained significant, which suggests that the prognostic power of these variables is similar. Using univariate analysis we proved that c-myc amplification and overexpression were highly significant for disease-free survival (DFS) (P = 0.0016 and P = 0.0001 respectively) and overall survival (OS) (P < 0.0001 and P = 0.0095 respectively), although by multivariate analysis c-myc overexpression was statistically significant only for DFS (P = 0.0001) and c-myc amplification only for OS (P = 0.0006). With regard to c-erbB-2, only its overexpression appeared to be significant for DFS and OS, although after multivariate analysis its prognostic power was weaker (P = 0.030 and P = 0.024 respectively). c-myc amplification and overexpression exhibited a tendency for locoregional recurrence (LRR) (P = 0.0024 and P = 0.0075 respectively), however, their prognostic value was lower after multivariate analysis and only CD remained significant.

Predictive value of c-erbB-2 and cathepsin-D for Greek breast cancer patients using univariate and multivariate analysis.

The value of various prognostic factors in breast cancer patients has been determined in a number of studies. One hundred thirty-eight Greek women were followed up over a 5-year period after surgery for breast cancer. Amplification and overexpression of c-erbB-2 was found in 22.4% and 29.7% of the respective cases, and the concentration of total cytosolic Cathepsin-D (CD) in 46.4% of them was high (> or = 60 pmol/mg protein). The examined biological variables were compared with standard clinicopathological prognostic factors for the disease and related to early relapse (ER; before 3 years), relapse-free survival (RFS; median, 5 years), and overall survival (OS; median, 5 years). It was found that high CD levels significantly shorten ER of both node-negative and node-positive patients (P < 0.0001 and P = 0.002, respectively) and have prognostic value for RFS and OS of node-negative patients (P = 0.0012 and P = 0.0288, respectively), but lose their value as relapse predictors for node-positive patients for periods longer than 3 years. Overexpression of c-erbB-2 was found to be predictive for OS of node-positive and -negative patients (P = 0.0048 and P = 0.0285, respectively), but its predictive power was weak for ER (P = 0.0456) and RFS (P = 0.0455) of node-negative patients and disappeared for node-positive patients. c-erbB-2 amplification offers minimal assistance to the prediction. In conclusion, high CD concentration is indicative of ER of patients, and c-erbB-2 overexpression correlates with OS of patients.

The polyadenylation inhibitor cordycepin (3'dA) causes a decline in c-MYC mRNA levels without affecting c-MYC protein levels.

Study of the distribution of the poly(A) tail length of c-myc mRNA in several cell lines revealed a distinct, prevailing population with short poly(A) tails, derived through sequential deadenylation. To elucidate the possible in vivo function of this distinct short tailed c-myc mRNA population, the polyadenylation inhibitor cordycepin was used. This resulted in a decline in steady state c-myc mRNA levels with the remaining messenger mostly oligoadenylated. However, c-MYC proteins did not follow the reduction of the c-myc mRNA. On the other hand, in cells exposed to physiological agents known to downregulate c-myc expression, the reduction of mRNA steady state levels, was reflected upon c-MYC protein levels. The dissociation between c-myc mRNA and protein levels caused by cordycepin was not due to the stabilization of the c-MYC proteins and was not an indiscriminate effect since in the presence of cordycepin, c-fos mRNA and protein levels concomitantly declined. Our data indicate that under these conditions, a long poly(A) tail is not instrumental for c-myc mRNA translation and furthermore, the discrepancy in the steady state of c-myc mRNA level: c-MYC protein ratio between control cells and cells treated with cordycepin indicates that c-myc mRNA is subjected to translational control.

Poly(A)polymerase activity levels in breast tumour cytosols.

The enzyme poly(A) polymerase (PAP) catalyses the polyadenylation of mRNA and its activity levels vary within the cell cycle. The levels of activity of this enzyme were measured in the cytosol of breast tumours from 62 untreated patients and compared to clinical prognostic parameters as well as other biological markers. The enzyme levels measured ranged from 3 to 46 units/mg protein. A statistically significant association was observed between high PAP activity values and the TNM stage of the disease as well as node invasiveness. Furthermore, there was a positive correlation between PAP activity values and c-erbB-2 overexpression but not with its amplification. No significant correlation was observed with c-myc amplification or overexpression and cathepsin D levels. A direct relationship between steroid receptor content and PAP activity levels, which was more prominent in the case of the progesterone receptor, was observed. However, also on the basis of previous data PAP activity may prove to be indicative of aggressive disease. Furthermore, measurements of PAP activity may contribute to the definition of the biological profile of tumour cells.

In vivo generation of 3' and 5' truncated species in the process of c-myc mRNA decay.

It has been demonstrated that the half-life of c-myc mRNA is modulated in response to physiological agents. The elucidation of the decay process and the identification of the critical steps in the in vivo c-myc mRNA degradation pathway can be approached by following the fate of c-myc mRNA under the influence of such factors. IFN-alpha was the factor used to modulate c-myc mRNA half-life in HeLa 1C5 cells, a stable clone derived from HeLa cells. This cell line carries multiple copies of the c-myc gene, under the control of the dexamethasone inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Exposure of HeLa 1C5 cells to IFN-alpha resulted in a further 2-fold increase over the dexamethasone-induced c-myc mRNA. However, the c-myc mRNA in IFN-alpha treated cells was less stable than that in the control cells. RNase H mapping of the 3' untranslated region of c-myc mRNA revealed, in addition to the full length mRNA, three smaller fragments. These fragments were proven to be truncated, non-adenylated c-myc mRNA species generated in vivo. Exposure of HeLa 1C5 cells to Interferon-alpha before induction with dexamethasone resulted in the enhanced presence of these intermediates. RNase H analysis of c-myc mRNA after actinomycin D chase revealed that deadenylation led to the formation of a relatively more stable oligoadenylated c-myc mRNA population which did not appear to be precursor to the truncated intermediates. The detection of truncated 3' end c-myc mRNA adenylated fragments as well, implies that the c-myc mRNA degradation process may follow an alternative pathway possibly involving endonucleolytic cleavage.

delta IK17: an antigen expressed on human lymphocytes.

Anti delta IK17 monoclonal antibody was produced by fusing SP2/0/Ag 14 myeloma with spleen cells of BALB/c mice immunized with normal human thymocytes. a delta IK17 antibody recognizes a 44kD cell surface protein detected on human lymphocytes. delta IK17 is expressed on human thymocytes, CD4+ and CD8+ T cell subsets, B, NK cells, as well as on activated cells. The antigen is detected on cells during the early, intermediate and late stages of lymphocyte maturation. In addition, the expression of the antigen is correlated with ontogenesis. A T+ delta IK17+ subpopulation responded poorly to TPA stimulation and provided a better helper signal for PWM-induced IgM synthesis than T+ delta IK17- cells. In addition, different levels of delta IK17 expression were detected in several hematological diseases tested.

delta IK17 antigen: a possible early marker of cancer development.

delta IK17 is a 44 kD molecule located on the surface of T, B and NK cells in normal peripheral blood mononuclear cells (PBMC) (1). The portion of PBMC expressing delta IK17 was determined in 52 patients with benign breast diseases, 182 patients with breast malignancies and 132 healthy individuals. The percentage of delta IK17-positive cells was significantly lower in the early stages (I-IIA) of malignancy compared to that of healthy donors. However, the percentage of PBMC expressing delta IK17 tended to increase as the stage of the disease advanced. delta IK17 seems to be the only antigen among the other cellular markers tested (CD2, CD4, CD8, HLA-DR) with a statistically significant correlation between a low percentage of positive cells and the early stages of malignancy and between a high expression and advanced disease. Its potential use as a tumor marker in breast cancer is discussed here.

c-erbB-2 overexpression may be used as an independent prognostic factor for breast cancer patients.

Insight into correlations between specific gene amplification and the clinical behavior of tumors may provide new prognostic tools High c-erbB-2 expression is an early feature in some breast tumors, whereas c-myc may be involved in the development of neoplasia. After an initial flurry of excitement about their prognostic significance, controversy has arisen about their independent importance. In an attempt to solve this problem, we decided to study c-erbB-2 and c-myc amplification and overexpression in 62 unselected breast carcinomas. This was done in order to correlate them statistically with one another, as well as with other prognostic parameters. A positive correlation was discovered between c-erbB-2 amplification and overexpression (P = 0.02); however, the correlations between c-erbB-2 amplification and c-myc amplification and overexpression (P = 0.06 and P = 0.095 respectively) were found to be negative. In addition, no correlation was found to exist between c-erbB-2 amplification and Cathepsin-D, steroid receptors, node status and menopausal status, as well as between c-erbB-2 overexpression and Cathepsin-D, node invasiveness, tumor status, grade or menopausal status. In conclusion, the c-erbB-2 overexpression has positive correlation with only a few other prognostic parameters, and therefore can be used as an independent prognostic factor.

Identification of a tripartite basal promoter which regulates human terminal deoxynucleotidyl transferase gene expression.

In order to locate the promoter region of the human terminal deoxynucleotidyl transferase gene, serially truncated segments of the 5'-flanking region of the gene were cloned into a chloramphenicol acetyltransferase reporter vector. Transient transfection analyses of the terminal transferase-reporter gene constructs identified the basal promoter region within -34 to +40 base pairs relative to the transcription start site. Three promoter elements were defined in this region. The primary element is within 34 base pairs upstream of the transcription start site. The CAP site is 62 base pairs upstream of the translation start site. The secondary element involves sequences around the transcription start site. The third is located 25 base pairs downstream from the initiation site (+25 to +40). This tripartite basal promoter was not tissue specific; similar patterns of promoter activity were observed in terminal transferase expressing and non-expressing cells. Transfection analyses also indicated the presence of negative regulatory elements upstream of the basal promoter region, and these elements were preferentially active in cells expressing terminal transferase.

Cathepsin-D and c-erb-B 2 have an additive prognostic value for breast cancer patients.

In breast cancer, axillary lymph node invasiveness is the major prognostic factor in predicting relapse and metastasis. Nevertheless, since 30% of node-negative tumours also relapse, it is necessary to develop other independent prognostic factors. Oncogene amplification and overexpression as well as the level of cathepsin-D have been proposed as additional prognostic factors. Recent studies suggest that the acidic lysosomal proteinase cath-D, present in all cells and known to be secreted in breast cancer cells, may be implicated in the process of tumour invasion and metastasis. We have compared the cytosolic cath-D level with the amplification and the overexpression of the oncogenes c-myc and c-erb-b-2 in 62 breast carcinomas (52 primary and 10 metastatic). Using a cut-off level of 60 pmol/mg protein, the status of cath-D showed a positive correlation with c-myc amplification (P = 0.01) or overexpression (P = 0.02). In contrast, no correlation was found between cath-D and c-erb-B-2 amplification or overexpression. Also, no correlation was found between cath-D and established prognostic factors such as node invasiveness, steroid receptors, grade and menopausal status. Nevertheless, a weak correlation was found between cath-D levels and tumour status (P = 0.05). In conclusion, in breast cancer, a high cytosolic cath-D concentration is more frequent in tumours with c-myc amplification and overexpression but is dissociated from c-erb-B-2 amplification or overexpression, suggesting that the determination of cath-D, as well as the latter two markers, will have an additional prognostic value.

Tissue-specific expression of human terminal deoxynucleotidyl transferase is regulated at the transcriptional level.

Transcriptional regulation of expression of the terminal deoxynucleotidyl transferase gene in normal thymus and in differentiation arrested cells was demonstrated by analyzing steady-state levels of TdT RNA as well as the relative transcription rate of the gene. Terminal transferase transcripts were detected only in those cells and tissues that contained antigen and enzyme activity. The relative rates of transcription correlated with levels of mRNA as well as with levels of the protein. These data suggest that expression of this gene in normal and leukemic cells is modulated at the level of transcription.

Patterns of adenosine deaminase, ecto-5'-nucleotidase, poly(A)polymerase and surface light chain expression in chronic lymphocytic leukemias.

The levels of activity of three enzymes have been measured in the circulating malignant lymphocytes of 47 patients with B chronic lymphocytic leukemia (CLL). These were the purine degradative enzymes, adenosine deaminase (ADA) and ecto-5'-nucleotidase (5'NT) and the enzyme responsible for the polyadenylation of mRNA, poly(A) polymerase. The patterns of activity of the above enzymes and the expression of surface immunoglobulin light chains were examined. A heterogeneity in the specific activity of the enzymes was observed which could not be attributed to variations of the percentage of B lymphocytes. A positive correlation was found between ADA and poly(A)polymerase activity (r = 0.383, p less than 0.01). Furthermore, the expression of immunoglobulin light chain phenotype was inversely related to 5'NT specific activity; CLL cases in which less than 20% of the cells expressed lambda chain phenotype, presented 5'NT specific activity of 16.7 +/- 3.3 (S.E.) nmol/h/10(6) cells, whereas in CLL cases with more than 20% of the cells expressing this phenotype the enzyme specific activity was 4.8 +/- 1.6 (S.E.) nmol/h/10(6) cells (p less than 0.02). These findings suggest that the simultaneous determination of enzymatic activities and immunological markers, might be useful in defining subsets in CLL and the subsequent clinical treatment.